Protein Folding
Proteins are synthesized in the cytoplasm, and the newly synthesized proteins are secreted into the lumen of the endoplasmic reticulum (ER) in a largely unfolded state. In general, protein folding is governed by the principle of self assembly. Newly synthesized polypeptides fold into their native conformation based on their amino acid sequences (Anfinsen et al., Adv. Protein Chem. 1975; 29:205-300). In vivo, protein folding is complicated, because the combination of ambient temperature and high protein concentration stimulates the process of aggregation, in which amino acids normally buried in the hydrophobic core interact with their neighbors non-specifically. To avoid this problem, protein folding is usually facilitated by a special group of proteins called molecular chaperones which prevent nascent polypeptide chains from aggregating, and bind to unfolded protein such that the protein refolds in the native conformation (Hartl, Nature 1996; 381:571-580).
Molecular chaperones are present in virtually all types of cells and in most cellular compartments. Some are involved in the transport of proteins and permit cells to survive under stresses such as heat shock and glucose starvation. Among the molecular chaperones (Gething et al., Nature 1992; 355:33-45; Caplan, Trends Cell. Biol. 1999; 9:262-268; Lin et al., Mol. Biol. Cell. 1993; 4:109-1119; Bergeron et al., Trends Biochem. Sci. 1994; 19:124-128), Bip (immunoglobulin heavy-chain binding protein, Grp78) is the best characterized chaperone of the ER (Haas, Curr. Top. Microbiol. Immunol. 1991; 167:71-82). Like other molecular chaperones, Bip interacts with many secretory and membrane proteins within the ER throughout their maturation, although the interaction is normally weak and short-lived when the folding proceeds smoothly. Once the native protein conformation is achieved, the molecular chaperone no longer interacts with the protein. Bip binding to a protein that fails to fold, assemble or be properly glycosylated, becomes stable, and is usually followed by degradation of the protein through the ER-associated degradation. This process serves as a “quality control” system in the ER, ensuring that only those properly folded and assembled proteins are transported out of the ER for further maturation, and improperly folded proteins are retained for subsequent degradation (Hurtley et al., Annu. Rev. Cell. Biol. 1989; 5:277-307).
Certain missense mutations result in amino acid substitutions that alter the native and proper folding of the protein. To correct these misfoldings, investigations have attempted to use various molecules as artificial chaperones. High concentrations of glycerol, dimethylsulfoxide, trimethylamine N-oxide, or deuterated water have been shown to stabilize the mutant protein and increase the intracellular trafficking of mutant protein in several diseases (Brown et al., Cell Stress Chaperones 1996; 1:117-125; Burrows et al., Proc. Natl. Acad. Sci. USA. 2000; 97:1796-801). These compounds are considered non-specific chemical chaperones to improve the general protein folding, although the mechanism of the function is still unknown. The high dosage of this class of compounds required for efficacy makes them difficult or inappropriate to use clinically, although they are useful for the biochemical examination of folding defect of a protein intracellularly. They also lack specificity.
Active Site Specific Chaperones for Enzymes
Co-owned U.S. Pat. Nos. 6,274,597, and 6,774,135 which are incorporated herein by reference, disclose a novel therapeutic strategy for Fabry disease, a lysosomal storage disorder (LSD) caused by a deficiency in lysosomal α-galactosidase A (α-Gal A) activity. α-Gal A deficiency often results from mutations in the gene that encode mutant proteins that result in folding defects. It was discovered that administration of 1-deoxygalactonojirimycin (DGJ), a potent competitive inhibitor of α-Gal A, effectively increased in vitro stability of a mutant α-Gal A (R301Q) at neutral pH. These results were also observed in lymphoblasts established from Fabry patients with the R301Q or Q279E mutations. Surprising, cultivation of the cells with DGJ at sub-inhibitory concentrations resulted in a substantial increase of residual enzyme activity. Furthermore, oral administration of DGJ to transgenic mice overexpressing a mutant (R301Q) α-Gal A substantially elevated the enzyme activity in major organs (Fan et al., Nat. Med. 1999; 5:112-115).
The principle of this strategy is as the follows. Since the mutant enzyme appears to fold improperly in the ER where pH is neutral, as evidenced by its instability at pH 7.0 in vitro (Ishii et al., Biochem. Biophys. Res. Comm. 1993; 197:1585-1589), the enzyme would be retarded in the normal transport pathway from the ER to the Golgi apparatus and subjected to rapid degradation. If a mutant enzyme could be efficiently transported to the lysosomes, it may retain normal or near normal kinetic properties and would remain active, because the mutant enzyme is sufficiently stable below pH 5.0. The goal, therefore, was to induce the mutant enzyme to adjust the proper conformation in the ER. In particular, a compound that can induce a stable molecular conformation of the enzyme could serve as an “active-site specific chaperone” (ASSC) or “pharmacological chaperone” to stabilize the mutant enzyme in a proper conformation for transport to the lysosomes. In the case of enzymes, such a compound unexpectedly was discovered to be a competitive inhibitor of the enzyme. Competitive inhibitors of an enzyme are known to occupy the catalytic site of the properly folded enzyme, resulting in stabilization of its correct conformation in vitro. It was found that they also serve as ASSCs or pharmacological chaperones to induce the proper folding of enzyme in vivo, thus rescuing the mutant enzyme from the ER quality control system.
Co-owned U.S. Pat. Nos. 6,583,158, 6,589,964, 6,599,919, and U.S. application Ser. No. 10/304,395 to Fan et al., exemplify the ASSC strategy with numerous other lysosomal storage diseases, including Gaucher disease. These findings demonstrate that this therapeutic strategy of using potent competitive inhibitors as ASSCs to increase the residual enzyme activity in the patient's cells is not limited to Fabry disease, and can be applied to enzyme deficiency diseases of this sort, and particularly to lysosomal storage disorders. In general, effective ASSCs of specific enzymes associated with particular diseases are potent competitive inhibitors of the enzyme. Unexpectedly, a more potent inhibitor of the enzyme acts as a better ASSC for the mutant enzyme (Fan, Trends Pharmacol Sci. 2003; 24:355-60).
Potent Inhibitors of β-glucocerebrosidase
β-Glucocerebrosidase (GCase, or acid β-glucosidase) is a lysosomal hydrolase that catalyzes the hydrolytic cleavage of glucose from glucosylceramide (Brady et al., Biochem. Biophys. Res. Commun. 1965; 18:221-225). The deficiency of the enzyme activity results in progressive accumulation of glucosylceramide, a normal intermediate in the catabolism of globoside and gangliosides, in lysosomes of macrophages, leading to Gaucher disease, the most common lysosomal storage disorder (Beutler et al., in The Metabolic and Molecular Bases of Inherited Disease, 8th ed., McGraw-Hill, New York 2001, 3635-3668).
Details regarding the disease and therapeutic treatment will be described herein below. Sawkar et al. have reported that the addition of an inhibitor of GCase to a fibroblast culture medium leads to a 2-fold increase in the activity of N370S GCase, indicating that a potent inhibitor of GCase may be of therapeutic interest in the treatment of Gaucher disease, although the particular inhibitor was not sufficient enough as a therapeutic agent because of high cytotoxocity (Sawkar A. R. et al., Proc Natl Acad Sci U S A. 2002; 99(24):15428-33). Therefore, effort has been taken to design and synthesize potent inhibitors for GCase.
The catalytic mechanism of β-glycosidases is believed to proceed via a covalent glycosyl-enzyme intermediate and positive charge generated at the anomeric position (Ichikawa et al., J. Am. Chem. Soc. 1998; 120:3007-3018; Heightman et al., 1999; Angew. Chem. Int. Ed. 1999; 38:750-770). Ichikawa et al. have designed a class of potent inhibitors for β-glycosidases, 1-N-iminosugars in which a nitrogen atom is at the anomeric position of a monosaccharide (Structure 1A, Isofagomine or hydroxy-piperidine). In a preliminary study, D-glucose-type 1-N-iminosugar (isofagomine, or hydroxypiperidine 1) has been shown to be a potent inhibitor of GCase (U.S. Pat. No. 6,583,158 to Fan et al.). N-alkyl derivatives of 1-deoxynojirimycin (DNJ) are also potent inhibitors of GCase, particularly those have longer alkyl group (greater than C6 alkyl chain), although DNJ itself and those N-alkyl DNJ with shorter chains are not inhibitory (Structure 1B, N-nonyl 1-deoxynojirimycin) (U.S. Pat. No. 6,583,158). However, these inhibitors are either not specific enough or not potent enough towards to the GCase and not suitable for the treatment of Gaucher disease. Based on these findings, it was realized that GCase may contain two substrate binding sites in the catalytic domain: one which recognizes the glucosyl residue; the other which recognizes the ceramide moiety (Structure 1C, 6-C-nonyl hydroxypiperidine, RD-1: recognition domain 1; RD-2: recognition domain 2.). Determination of the crystal structure of GCase revealed an annulus of hydrophobic residues surrounding the entrance to the monosaccharide binding site (Dvir et al., EMBO reports 2003; 4:1-6), suggesting that a hydrophobic moiety attached to a sugar residue with a long alkyl chain is required for the interaction to the hydrophobic amino acid residues.

Thus, there remains a need in the art to design or identify specific competitive inhibitors of enzymes, and evaluate them for their ability to act as chaperones for the corresponding mutant enzymes that are associated with numerous LSDs, particularly inhibitors of GCase associated with Gaucher disease, and other disorders resulting from misfolded proteins.